Single‐cell transcriptomics stratifies organoid models of metabolic dysfunction‐associated steatotic liver disease

Abstract Metabolic dysfunction‐associated steatotic liver disease (MASLD) is a growing cause of morbidity with limited treatment options. Thus, accurate in vitro systems to test new therapies are indispensable. While recently, human liver organoid models have emerged to assess steatotic liver disease, a systematic evaluation of their translational potential is still missing. Here, we evaluated human liver organoid models of MASLD, comparatively testing disease induction in three conditions: oleic acid, palmitic acid, and TGF‐β1. Through single‐cell analyses, we find that all three models induce inflammatory signatures, but only TGF‐β1 promotes collagen production, fibrosis, and hepatic stellate cell expansion. In striking contrast, oleic acid ameliorates fibrotic signatures and reduces the hepatic stellate cell population. Linking data from each model to gene expression signatures associated with MASLD disease progression further demonstrates that palmitic acid and TGF‐β1 more robustly model inflammation and fibrosis. Our findings highlight the importance of stratifying MASLD organoid models by signatures of clinical disease progression, provide a single‐cell reference to benchmark future organoid injury models, and allow us to study evolving steatohepatitis, fibrosis, and HSC susceptibility to injury in a dynamic, multi‐lineage human in vitro system.

d. UMAP plot from a., showing the cell type annotations generated by the CellTypist 1 classifier.CellTypist was trained on cells from human liver scRNA-seq data 2 .Most probable matches for each cell type are displayed in the legend, if more than one cell type matches the query sample, alternate labels are separated by the "|" symbol.
e. Barplot indicating the replicate distribution as the proportion of total cells per cluster calculated with the Leiden algorithm at resolution 0.1 and ScType 3 database.
f. Barplots showing cell type distributions across replicates as the proportion of each cell type per total cells per sample.Annotation performed with ScType 3 database followed by statistical enrichment with GSEA-py enrichr 4 .
g. Lineplots showing cluster robustness metrics (Davies Bouldin index and Silhoutte score) in dependence to number of clusters generated through increasing Leiden resolutions (0.1 -1) for OS-and ULA-HLO scRNAseq data.Leiden resolution 0.1 generating four clusters in each context was chosen for subsequent analysis.Silhouette score multiplied by 10 for joint visualization.
h. ForceAtlas2 representations of the cells from OS-and ULA-HLOs, colored by the mean scaled expression of HSC marker genes, corresponding to Fig. 2d.

h.
ForceAtlas2 plots mapping cells from OS-HLOs treated with OA (500 µM, n = 2), PA (500 µM, n = 2), TGF-β1 (10 ng/ml, n = 2), and their respective controls (n = 8) colored by replicate (left) and cell cycle phase (right).b.Barplot indicating the replicate distribution as the proportion of total cells per cluster for each cluster.Cell clusters are colored in the order of their appearance.AH -adult hepatocyte-like, CHOL -cholangiocyte-like, DCs -ductal cell-like, FH1 -fetal hepatocyte 1-like, cAH -cycling adult hepatocyte-like, FIB -fibroblast-like, HB1 -hepatoblast 1-like, HB2 -hepatoblast 2-like, HSCs -hepatic stellate cell-like, SMCs -smooth muscle cell-like.c.ForceAtlas2 plots from a., showing the scaled expression of canonical marker genes.d.Matrixplot shows the scaled mean expression for marker genes in cholangiocyte sub-clusters.Canonical marker genes (bottom) are sorted by cell type (top).Hierarchical clustering is represented by the dendrogram on the right.e. Barplots showing cell type distributions across replicates as the proportion of each cell type per total cells per sample.Cell clusters are colored in the order of their appearance.f.Barplots showing zonal distributions (Methods) across replicates as the proportion of each cell type per total cells per sample for adult-hepatocyte like cells.Zonation categories are colored in the order of their appearance.g.Representative qRT-PCR results for COL1A1 and DES in two PSC lines.Shown are relative mRNA levels normalized to ACTB (2 ddCt ⁻ ).T-test (two-tailed), p-values as indicated in the figure, ns, not significant.Data are shown for one experiment where each dot represents one well of HLOs.Data are representative for 3 -4 experiments performed on HLOs differentiated from each PSC line.Summary of all qRT-PCR experiments for COL1A1 and DES in two PSC lines.Shown are relative mRNA levels normalized to ACTB (2 ddCt ⁻ Fig. S6